Metabolites of Key Flavor Compound 2,3,5-Trimethylpyrazine in Human Urine

Pyrazines are among the most important compound class conveying the odor impressions “roasty”, “nutty”, and “earthy”. They are formed by the Maillard reaction and occur ubiquitously in heated foods. The excretion of metabolites of the key flavor odorant 2,3,5-trimethylpyrazine, abundant in the volatile fraction of roasted coffee, was investigated. Based on literature suggestions, putative phase 1 and phase 2 metabolites were synthesized, characterized by nuclear magnetic resonance and mass spectroscopy data and used as standards for targeted, quantitative analysis of coffee drinkers’ urine using stable-isotope-dilution-ultrahigh-performance liquid chromatography tandem mass spectroscopy (SIDA-UHPLC–MS/MS). The analysis of spot urine samples from a coffee intervention study revealed 3,6-dimethylpyrazine-2-carboxylic acid, 3,5-dimethylpyrazine-2-carboxylic acid, and 5,6-dimethylpyrazine-2-carboxylic acid were quantitatively dominating metabolites. Only negligible traces of pyrazinemethanols (3,6-dimethyl-2-pyrazinemethanol and 3,5,6-trimethylpyrazine-2-ol), glucuronides ((3,6-dimethylpyrazine-2-yl-)methyl-O-β-D-glucuronide and (3,5-dimethylpyrazine-2-yl-)methyl-O-β-D-glucuronide), and sulfates ((3,6-dimethylpyrazine-2-yl-)methyl-sulfate and (3,5-dimethylpyrazine-2-yl-)methyl-sulfate) were detected.


INTRODUCTION
Pyrazines, a compound class of aromatic heterocycles with nitrogen in the 1-and 4 position, are considered one of the most important compound class conveying earthy, nutty, and roasty odors. 1,2Pyrazines are common in heated foods, like roasted meat, bread, cocoa, or coffee, 3,4 as they are naturally formed during nonenzymatic browning (Maillard) reaction from amino acids and reducing sugars upon heating.This reaction takes place in both aqueous and dry systems at elevated temperature, 5 even under physiological conditions. 6yrazines in foods can also be products of microbial origin, for example, during fermentation processes. 4Pyrazines have low odor thresholds and are among the compounds used in the food industry as flavor ingredients with GRAS status (generally recognized as safe) confirmed by the FEMA (Flavor Extract Manufacturing Association). 2 Despite their broad abundance in foods, 3,4 literature on uptake, metabolism, and excretion of dietary alkyl-substituted pyrazines is limited and mainly comprises data from animal experiments. 2Reported pathways primarily involve cytochrome P-450-catalyzed oxidation of ring-substituted alkyl groups to form primary alcohols and subsequently carboxylic acids, while ring-hydroxylation apparently only occurs for selected pyrazines.It has been suggested that phase 1 metabolites formed by oxidation and hydroxylation may further be excreted in the urine after conjugation with glucuronides and sulfates because these are among the most important metabolic detoxication routes. 7However, sound literature in humans is lacking. 2,4,8sides socializing aspects, the roasty aroma and the bitter taste of coffee brew is one of the key drivers for its consumption.Pyrazines substantially contribute to the aroma of coffee, 9−11 and total pyrazine concentrations of 82.1−211.6 mg/kg have been reported in commercial roast coffee powder.The derivative 2-methylpyrazine was of highest abundance followed by 2,5-dimethylpyrazine and 2,6-dimethylpyrazine. 12 Pyrazines are effectively extracted into the brew during coffee making, with extraction rates reaching 82%. 13A recent human coffee intervention study reported that the pyrazines 2methylpyrazine, 2,5-dimethylpyrazine, and 2,6-dimethylpyrazine in coffee were metabolized into the corresponding 2carboxylic acid derivatives and excreted via the urine. 14In coffee, the roasty smelling 2,3,5-trimethylpyrazine (TMP, 1) occurs in concentrations between 1 and 6.7 mg/kg and has an odor threshold of ∼50 ng/L air. 12,15he focus of the present study was the key flavor compound TMP.Based on the available information, it was the aim to synthesize putative phase 1 and 2 metabolites of TMP (Figure 1) in order to quantitate excreted metabolites of TMP in human urine.d 3 ,5-dimethylpyrazine-2-yl-)methyl-sulfate (d 3 -5b) were synthesized (refer to next chapter 2.1).
2.4.1.(3,6-Dimethylpyrazine-2-yl-)methyl-sulfate (5a).6.9 mg, yield = 16%. 12 The solution was cooled to 0 °C and a solution of methyl-d 3 -magnesium iodide (1 M in ether, 200 mL, 200 mmol) was added dropwise.The mixture was stirred overnight and carefully quenched with diluted hydrochloric acid (1 M, 100 mL).The organic layer was dried with anhydrous sodium sulfate and concentrated.The crude product was purified on silica gel (pentane/ether 7:3) to afford the title compound (3.86 g, yield = 22%). 1H NMR (CD 3 Cl, 500 MHz): δ ppm 8.16 (s, 1 H), 2.50 (s, 3 H), 2.48 (s, 3 H). 13 d 3 -4b). 1  In brief, the study participants went through a run-in phase (3 days) in which they were asked to refrain from the consumption of coffee, coffee-containing foods/sweets, caffeine-containing foods/ drinks, and strongly flavored meals.The study phase followed immediately after and comprised three consecutive days.On each study day, the volunteers fasted for 10−15 h prior to admittance to the study center.A morning spot urine sample (sampling points t1, t3, t5) was collected.Then, either tap water (day one, control, 250 mL), a dose of coffee brew (day two, 250 mL), or two doses of coffee brew (day three, 500 mL) was consumed.Further spot urine samples were collected after 4 h (day one and two, time point t2 and t4) and 2 h (day three, t6; Figure 2).The administered coffee brew was freshly prepared as follows: whole roasted coffee beans (50 g, Tchibo CaffeC rema, 100% Arabica) were ground in a coffee mill (10 s) and passed through a sieve (2 mm).The ground material (48.75 g) was weighed into a French press, mixed with freshly boiled table water (Evian, 750 mL), and incubated (4 min).Finally, the press was passed through the suspension and the resulting brew (200 and 400 mL, respectively) was poured into cups.Aliquots were used for quantification of 2,3,5trimethylpyrazine (1).The coffee brew contained 4.08 μM (±9.2%, n = 3) of compound 1 determined by stable isotope dilution analysis (GC−MS/MS) and an in-house protocol.One dose of coffee (200 mL, day 2) delivered 0.82 μmol and two doses of coffee (400 mL day 3) delivered 1.63 μmol of compound 1.

Quantitative Analysis (UHPLC−MS/MS). 2.7.1. Instrumentation.
The UHPLC−MS/MS system consisted of a QTrap 6500+ MS/MS system (Sciex, Darmstadt, Germany) connected to an ExionLC UHPLC system (Sciex, Darmstadt, Germany).The MS instrument was operated in the ESI + mode, applying a spray voltage of +5500 V and a source temperature of 600 °C.The nebulizer gas was set to 55 psi, the heating gas was set to 65 psi, and the curtain gas (nitrogen) was set to 35 psi.The MS/MS parameters, including the collision cell entrance potential (CEP), declustering potential (DP), collision energy (CE), and cell exit potential (CXP), were tuned for each individual compound and mass transition.For the chromatographic separation, a gradient elution at 50 °C on a biphenyl column (Kinetex Biphenyl, 150 × 2.1 mm, 1.7 μm, Phenomenex, Aschaffenburg, Germany) was performed using eluent A (0.1% formic acid, 1 mM NH 4 Ac in water), eluent B (0.1% formic acid, 1 mM NH 4 Ac in acetonitrile), and following gradient at a flow rate of 0.5 mL/min: 0 min 1% B, 2 min 5% B, 8 min 3% B, 9 min 50% B, 11 min 50% B, 11.5 min 1% B, and 15 min 1% B. The flow was directed to the MS from minute 1 to 8.
Journal of Agricultural and Food Chemistry 6.3 min 60% B, 7 min 1% B, and 10 min 1% B. The injection volume was 5 μL.The flow was directed to the MS from minute 1 to 5.

Stock Solutions and Calibration.
Individual stock solutions of the synthesized reference compounds and the internal standards were prepared in deuterated methanol (d 4 -methanol) (range 8−34 mM determined by quantitative 1 H NMR, 21 ).Aliquots were subsequently combined and diluted in 50% aqueous ACN to prepare one separate mixture of reference compounds (mixed analyte solution) with a final concentration of 100 μM per compound and another separate mixture of internal standards (mixed IS solution) with a final concentration of 10 μM per compound.Artificial urine (AU 16 ) was used as the matrix for calibration standards and quality controls.The mixed analyte stock was serially diluted with AU (1 + 1, v/v) to obtain dilutions from 1250 to 9 nM.Quality control samples (QCs) were prepared in AU in triplicates (n = 3) at 313 nM and analyzed in replicates (n = 6) to assess precision and accuracy.For the sulfates, calibration standards and QC samples were run in replicates (n = 5) (Table 1).

Sample
Preparation.An aliquot of the standard or authentic sample (urine), respectively (450 μL), was spiked with the mixed IS solution (50 μL) and transferred to an autosampler vial.Aliquots (5 μL) were injected into the UHPLC−MS/MS system.Calibration curves were established by plotting area ratios of the analyte and internal standard versus the respective concentration ratios.Calibration curves were 1/x weighed and had R 2 > 0.99.2.7.4.Data Processing.Processing of raw data and calculation of quantitative data, calibration curves, and QC statistics was done in analyst 1.6.3(Sciex, Darmstadt, Germany).Quantitative data were analyzed with GraphPad 9.3.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com).Data were tested for outliers and normality/lognormality with the Kolmogorov−Smirnov test.One-way analysis of variance (ANOVA) was done with log-

RESULTS AND DISCUSSION
Pyrazines are frequently consumed with diet as they are common odorants in thermally processed foods like toasted bread or roasted coffee. 3Animal experiments indicate that metabolism primarily involves oxidation and hydroxylation leading to carboxylic acid derivatives as final excretion products.The excretion of phase 2 metabolites has been hypothesized by Adams et al. and Muller and Rappert. 2,8In contrast to dimethylpyrazines, little is known about the key flavor odorant TMP.
Aiming at the clarification of excreted metabolites formed from TMP, we synthesized phase 1 and phase 2 metabolites based on literature suggestions 2,4,8 and one additional deuterium-substituted derivative for each class of analyte to serve as the internal standard.We developed stable isotope dilution assays (SIDAs) to analyze spot urine samples, collected from a human coffee intervention study, by means of UHPLC−MS/MS.The compounds were individually introduced into the mass spectrometer via a syringe for software-assisted tuning of ion sources and path parameters, as well as optimization of energies for collision-induced dissociation (CID) to enable detection in the multiple reaction monitoring (MRM) mode.Glucuronides, pyrazinemethanols, and pyrazine-2-carboxylic acids were easily tuned in positive electrospray (ESI + ).Chromatographic separation was achieved on a 150 mm biphenyl column with acetonitrile and water, each containing 0.1% formic acid and 1 mM ammonium acetate as modifiers.Calibration standards, quality controls, and urine samples were analyzed with a method in positive electrospray for the analytes 2−4 and a second method in negative electrospray for the sulfated analytes.Because it was impossible to obtain analytefree human urine due to the broad occurrence of the odorant 2,3,5-trimethylpyrazine in foods, we used AU prepared from inorganic and organic salts, creatinine, citric acid, and urea to prepare calibration standards and QC. 16Area ratios of analyte versus internal standard were plotted against the concentration ratios.The calibrated range was between 9 and 1250 nM, with precision of back-calculated standards <15% and accuracy between 91.7 and 112.6%.QCs showed a precision <13.2% and accuracy between 94.5 and 109.2%(Table 1 and supporting Table 1).
To investigate the contribution of roast coffee consumption on the excretion of metabolites formed from dietary TMP under typical house-hold/real-life conditions, we obtained urine samples from a pilot coffee drinking study recently conducted in our institute.In brief, six healthy participants (3m, 3f) abstained from roast coffee consumption for three consecutive days to reduce internal levels of TMP metabolites.On study day one, the participants brought a morning spot urine sample.Tap water was consumed and a second spot urine sample was collected after 4 h.The following day, the procedure was repeated but instead of tap water, a serving of roast coffee brew (200 mL) was consumed and urine collected.On study day three, the procedure was repeated but instead of one serving of roast coffee brew, two servings were consumed (400 mL) and a final spot urine sample was collected after 2 h.The concentration of TMP in the roast coffee brew determined by SIDA-GC−MS/MS was 4.08 μM (±9.2%, n = 3), the ingested amount therefore was 0.82 μmol on day 2 (one coffee serving), and 1.63 μmol on day 3 (two servings).The collected spot urine samples were spiked with the internal standards and analyzed by UHPLC−MS/MS.Concentrations were not adjusted for creatinine as the primary aim of the study was to identify and quantitate key metabolites of TMP rather than to do a quantitative recovery of ingested amounts.
From the selection of metabolite candidates, only the carboxylic acids 3a−c were in the quantifiable concentration range and showed substantial abundance (Figure 3).In contrast, hydroxylated TMP derivatives 2a−d were either not detectable (<LoD) or not quantifiable (<LloQ).Phase 2 metabolites 4a−c were detected in some of the spot urines, but concentrations were below the LloQ.The sulfated analytes 5a and b were detected in all samples, but the concentration was below the LloQ, resulting in negligible concentrations compared to 3a−c.The derivative 5c was not detected.
Pyrazines are odorants in thermally processed foods, like bread crust, roasted meat, and coffee. 3Notably, abstinence from roasted coffee alone did not result in the absence of TMP-related metabolites in the morning urines, underlining the broad occurrence in foods.However, after consumption of roast coffee brew, the concentrations in excreted urine significantly rose for 3,6-dimethylpyrazine-2-carboxylic acid (3a) and 3,5-dimethylpyrazine-2-carboxylic acid (3b).This suggested roasted coffee is a substantial dietary source for Journal of Agricultural and Food Chemistry TMP, and the two compounds 3a and 3b were the major metabolites formed, being excreted in concentrations of >700 nM (Figure 3).Compared to these compounds, the detected traces (<9 nM) of phase 1 metabolites 2a−d and phase 2 metabolites 4a, 4b, and 5a/b were negligible, being either below the LoD or LloQ.
In conclusion, we report the application of a quantitative UHPLC−MS/MS-based stable isotope dilution assay and results from urine analysis of metabolites of TMP delivered by real-life doses of roasted coffee brew.−24 In contrast to untargeted metabolomics utilizing high-resolution MS-screening aiming at biomarker discovery, 25−27 the number of participants in the study the biosamples were available from was relatively small with six participants, and substantial interindividual differences in excreted concentrations were detected.Our targeted approach nevertheless succeeded in unambiguous identification and quantification of TMP metabolites as the data show that consumption of roasted coffee brew leads to elevated concentrations of TMP metabolites 3a and 3b.However, it is apparent that coffee is not the only contributor to their abundance.
Future pharmacokinetic investigations on the formation of carboxylated TMP metabolites will benefit from the study participants following a strict pyrazine-free diet during the runin period, taking noncoffee sources for TMP into account, to minimize initial metabolite abundance.Normalizing determined metabolite concentrations to individual creatinine concentrations will further help minimize the spread in the analytical data.Despite these limitations, the data suggest that the formation of phase 1 metabolites 3a−c was the preferred metabolization route for of TMP, being in line with previous results on other pyrazine derivatives, 14 and no substantial abundance of phase 2 metabolites was recorded.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.2c06418.Supporting InformationPrecision and accuracy of the analysis of putative metabolites of 2,3,5-trimethylpyrazine in quality controls (supporting Table 1); concentrations (means ± standard deviation) in human spot urine (supporting

Figure 2 )
aimed at the identification of odor-active coffee-derived compounds in biosamples.It was approved by the ethical committee of the Faculty of Medicine at the Technical University Munich, ethical vote 357/20 S. The study was registered in "Deutsches Register Klinischer Studien", DRKS00024380.The study protocol adhered to the Declaration of Helsinki for Human Intervention/clinical studies.Six healthy, nonsmoking participants with neither acute nor chronic diseases, no current medication, and no known orosensory disorders (m/w 3/3, age 25−43) participated in the study and gave written informed consent.

Figure 2 .a
Figure 2. Design of the pilot coffee drinking study to investigate TMP metabolites in human urine.
The pyrazinemethanols 2a−d produced intense [M + H] + pseudomolecular ions and generated mainly unspecific fragments with the loss water being the dominant one.The carboxylates (3a−c) ionized well in positive electrospray despite the acidic function.Main fragments were [M − H 2 O + H] + and the cleavage of the carboxylic acid group as a loss of formic acid [M − HCOOH + H] + .The glucuronide conjugates (4a−c) gave abundant [M + H] + pseudomolecular ions in ESI + and delivered daughter ions, indicating the loss of hexuronic acid and a further loss of water.With exception of the glucuronides, we noticed that the MRM traces were relatively noisy despite the two MS-filtering steps in Q1 and Q3.The sulfates 5a−c were tuned in negative electrospray.The two obtained fragments (m/z 80 and 96) indicated cleavage of the sulfate group.
2.6.Coffee Intervention Study.Urine samples were from a recent pilot intervention study (cf.